Advisor(s)

John R. Engen

Contributor(s)

David E. Budil, Todd Miller

Date of Award

2010

Date Accepted

12-2010

Degree Grantor

Northeastern University

Degree Level

M.S.

Degree Name

Master of Science

Department or Academic Unit

College of Arts and Sciences. Department of Chemistry and Chemical Biology

Keywords

analytical chemistry, Breast Tumor Kinase, hydrogen exchange mass spectrometry, SH2 domain

Subject Categories

Protein-tyrosine kinase, Breast--Tumors

Disciplines

Chemistry | Organic Chemistry

Abstract

Breast tumor kinase (Brk) is a non-receptor protein tyrosine kinase (NRPTK) that is overexpressed in 2/3 of breast tumors. Brk belongs to a small and highly uncharacterized family of NRPTKs, the Frk family. Despite its segregation on the NRPTK family tree based on genetic and sequential criteria, marked structural similarities of Brk to proteins of the Src family of NRPTKs suggest that Brk may share important regulatory features with these proteins. Brk is composed of an SH3, SH2, and catalytic domain, an arrangement similar to that of Src family proteins. This anomalous architecture leads to the proposition that Brk, like the Src proteins, may be autoinhibited by intramolecular interactions involving these important strucutral facets. For instance, it is thus hypothesized that Brk may exhibit the same dominant stabilizing interaction of its SH2 domain with its phosphorylated C-terminal tail, an interaction likened to that of an electrical socket and plug (respectively), as a means of rigidly locking the protein in its inactive/downregulated state. In order to confirm this suspected regulatory pathway, information crucial to designing specific inhibitors of oncogenic Brk, the Brk SH2 domain was expressed recombinantly and subjected to conformational studies by hydrogen exchange mass spectrometry. Active (free BrkSH2 domain) and inactive (BrkSH2 domain bound to a phosphorylated peptide) protein forms were emulated by selectively using phosphopeptide constructs during sample preparation. The outcome of hydrogen exchange experiments with Brk demonstrate little to no structural dynamics influenced by phosphopeptide binding. Minimal structural changes were detected using both a physiological phosphopeptide construct and a high-affinity phosphopeptide. These data are not consistent with similar hydrogen exchange studies utilizing a Src family kinase, which may suggest Brk undergoes regulation through an alternate pathway.

Document Type

Master's Thesis

Rights Holder

Kelly A. Reiser


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